Diagnostic reagents made by attaching cytidine containing nucleic acid probes to amino functionalized solid supports by bisulfite mediated transamination

ABSTRACT

This invention encompasses diagnostic reagents comprising a polynucleic acid probe having a specific binding sequence and having one or more cytidines outside of the specific binding sequence. The polynucleic acid probe is bound to an amino functionalized solid support by a bisulfite mediated transamination between a cytidine and an amino group on the solid support. When the specific binding sequence contains cytidine, the cytidine is replaced with 5-methylcytidine which has essentially the same binding capacity and yet does not participate in transamination reactions. The invention encompasses compositions of the polynucleic acid probe as well as test kits including the above identified reagent and assays utilizing the above reagent. This invention is useful in detecting viruses, fungi and bacteria in test samples such as body fluids and food samples as well as detecting DNA or RNA sequences in mammalian cells.

BACKGROUND OF THE INVENTION

This invention is in the field of diagnostic reagents and methods fordetecting deoxyribonucleic (DNA) or ribonucleic acid (RNA) sequences intest samples. The invention relates to the field of binding DNA to asolid support.

The present invention encompasses methods, reagents, compositions, andkits for detecting deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)from test samples. Embodiments of the present invention provide methodsfor rapid, sensitive detection of nucleic acid targets in food orclinical samples such as body fluids which contain viruses, or bacteria.These reagents and methods are adaptable to non-radioactive labelingtechniques and automation and are equally applicable to other DNA andRNA containing cells such as mammalian cells and fungi.

The term "polynucleic acid probe" refers to a nucleic acid sequencehaving a specific binding sequence or region and one or more cytidinesgenerally a polycytidine region wherein cytidines in the specificbinding sequence are replaced with 5-methylcytidine. The specificbinding sequence may be directly bindable to the nucleic acid sequenceto be determined or it may be bindable to a nucleic acid sequence boundto the sequence to be determined. The specific binding sequence may becomplementary to nucleic acid sequences of variable nucleic acidresidues or it may be complementary to homopolymer segments such aspolyadenosine, polyguanosine, polythymidine, or poly-5-methylcytidine.The polycytidine region of the polynucleic acid probe may be at the endof the probe or it may be centrally located with nucleic acid sequenceson either side, for example, a polycytidine region with two specificbinding sequences on either side.

The term "label" refers to a molecular moiety capable of detectionincluding, by way of example, without limitation radioactive isotopes,enzymes, luminescent agents and dyes The term "agent" is used in a broadsense, including any molecular moiety which participates in reactionswhich lead to a detectable response Also biological detecting systemsmay be employed e.g., bacteriophage oligonucleotide conjugates.

The term "retrievable" is used in a broad sense to describe an entitywhich can be substantially dispersed within a medium and removed orseparated from the medium by immobilization, filtering, partitioning, orthe like.

The term "support" when used includes conventional supports such asnitrocellulose or nylon filters, membranes, beads, and dip sticks andmicrotiter plate wells, as well as retrievable magnetic supports. Asolid support is a separation medium by which nucleic acid sequences canbe selectively bound and then separated from other components of thereaction mixture.

Genetic information is contained in living cells in threadlike moleculesof DNA. In vivo, the DNA molecule is a double helix each strand of whichis a chain of nucleotides. Each nucleotide is characterized by one offour bases: adenine (A), guanine (G), thymine (T). and cytosine (C). Thebases are complementary in the sense that, due to the orientation offunctional groups, certain base pairs attract and bond to each otherthrough hydrogen bonding. Adenine in one strand of DNA pairs withthymine in an opposing complementary strand. Guanine in one strand ofDNA pairs with cytosine in an opposing complementary strand. In RNA, thethymine base is replaced by uracil (U) which pairs with adenine in anopposing complementary strand.

DNA consists of covalently linked chains of deoxyribonucleotides and RNAconsists of covalently linked chains of ribonucleotides. The geneticcode of a living organism is carried upon the DNA strand in the sequenceof the base pairs.

Each nucleic acid is linked by a phosphodiester bridge between the fiveprime hydroxyl group of the sugar of one nucleotide and the three primehydroxyl group of the sugar of an adjacent nucleotide. Each linearstrand of naturally occurring DNA or RNA has one terminal end having a5'-hydroxyl group and another terminal end having a 3'-hydroxyl group.The terminal ends of polynucleotides are often referred to as being5'-terminal or 3'-terminal in reference to the respective free hydroxylgroup. Complementary strands of DNA and RNA form antiparallel complexesin which the 3'-terminal end of one strand is oriented and bound to the5'-terminal end of the opposing strand.

Nucleic acid hybridization assays are based on the tendency of twonucleic acid strands to pair at complementary regions. Presently,nucleic acid hybridization assays are primarily used to detect andidentify unique DNA or RNA base sequences or specific genes in acomplete DNA molecule, in mixtures of nucleic acid, or in mixtures ofnucleic acid fragments.

The identification of unique DNA or RNA sequences or specific geneswithin the total DNA or RNA extracted from tissue or culture samples mayindicate the presence of physiological or pathological conditions. Inparticular, the identification of unique DNA or RNA sequences orspecific genes, within the total DNA or RNA extracted from human oranimal tissue, may indicate the presence of genetic diseases orconditions such as sickle cell anemia, tissue compatibility cancer andprecancerous states, or bacterial or viral infections The identificationof unique DNA or RNA sequences or specific genes within the total DNA orRNA extracted from bacterial cultures or tissue containing bacteria mayindicate the presence of antibiotic resistance, toxins, viruses, orplasmids, or provide identification between types of bacteria.

Thus, nucleic acid hybridization assays have great potential in thediagnosis and detection of disease Further potential exists inagriculture and food processing where nucleic acid hybridization assaysmay be used to detect plant pathogenesis or toxin-producing bacteria.

One of the most widely used nucleic acid hybridization assay proceduresis known as the Southern blot filter hybridization method or simply, theSouthern procedure (Southern, E., J. Mol. Biol. I., 98, 503, (1975)).The Southern procedure is used to identify target DNA or RNA sequences.This procedure is generally carried out by immobilizing sample RNA orDNA to nitrocellulose sheets as the solid support. The immobilizedsample RNA or DNA is contacted with radio-labeled probe strands of DNAhaving a base sequence complementary to the target sequence carrying aradioactive moiety which can be detected. Hybridization between theprobe and the sample DNA is allowed to take place.

The hybridization process is generally very specific. The labeled probewill not combine with sample DNA or RNA if the two nucleotide entitiesdo not share substantial complementary base pair organization.Hybridization can take from three to 48 hours depending on givenconditions.

However, as a practical matter there is always non-specific binding ofthe labeled probe to supports which appears as "background noise" ondetection. Background noise reduces the sensitivity of an assay.Unhybridized DNA probe is subsequently washed away. The nitrocellulosesheet is placed on a sheet of X-ray film and allowed to expose. TheX-ray film is developed with the exposed areas of the film identifyingDNA fragments which have been hybridized to the DNA probe and thereforehave the base pair sequence of interest.

The use of radioactive labeling agents in conjunction with Southernassay techniques have allowed the application of nucleic acid assays toclinical samples. Radioactive decay is detectable even in clinicalsamples containing extraneous proteinaceous and organic material.However, the presence of extraneous proteinaceous and organic materialmay contribute to nonspecific binding of the probe to the solid support.Moreover, the use of radioactive labeling techniques requires a longexposure time to visualize bands on X-ray film A typical Southernprocedure may require 1 to 7 days for exposure. The use of radioactivelabeling agents further requires special laboratory procedures andlicenses.

The above problems associated with assays involving radioisotopic labelshave led to the development of techniques employing nonisotopic labels.Examples of nonisotopic labels include enzymes, luminescent agents, dyesand biological detecting systems such as bacteriophage. Luminescentlabels emit light upon excitation by an external energy source and maybe grouped into categories dependent upon the source of the excitingenergy, including: radioluminescent labels deriving energy from highenergy particles; chemiluminescent labels which obtain energy fromchemical reactions; bioluminescent labels wherein the exciting energy isapplied in a biological system; and photoluminescent or fluorescentlabels which are excitable by units of electromagnetic radiation(photons) of infrared, visual or ultraviolet light See, generally, Smithet al., Ann. Clin. Biochem., 18:253, 274 (1981).

Nonisotopic assay techniques employing labels excitable bynonradioactive energy sources avoid the health hazards and licensingproblems encountered with radioisotopic label assay techniques.Moreover, nonisotopic assay techniques hold promise for rapid detectionavoiding the long exposure time associated with the use of X-ray film.

However, nonisotopic assays have not conveyed the sensitivity orspecificity to assay procedures necessary to be considered reliable. Inluminescent assays, the presence of proteins and other molecules carriedin biological samples may cause scattering of the exciting light or mayabsorb light in the spectrum of emission of the luminescent label,resulting in a quenching of the luminescent probe.

In enzymatic assays, the presence of proteins and other moleculescarried in biological samples may interfere with the activity of theenzyme.

Similarly, in colorimetric assays, the change in color may not bedetectable over proteins and other materials carried in biologicalsamples.

The use of polynucleic acid probes in diagnostic assays for DNA and RNAis extensively taught in the prior art.

U.S. Pat. No. 4,358,535 extensively discusses the detection of variouspathogens using labeled polynucleotide probes. Thus probes,hybridization conditions and methods for detecting labeled probes arewell known.

U.S. Pat. No. 4,689,295 describes a test for salmonella using probesspecific for salmonella DNA This patent describes in detail theculturing, lysing, and sample preparation techniques. This patent alsodescribes fixing DNA to nitrocellulose filters.

U.S. Pat. No. 4,626,501, EPA 259,186 and EPA 251,527 describe DNA probeassays on solid supports.

PCT/US 86/01280 application describes various configurations of nucleicacid hybridization assays widely applicable to detecting nucleic acidsequences specific to various bacteria and viruses.

U.S. Pat. No. 4,139,346 describes nucleic acid probes covalently boundto a support paper modified with aminobenzyloxymethyl groups.

U.K. Patent Application GB 2169403A describes the formation of a complexwhere the DNA or RNA to be detected is bound to two probes in differentcomplementary regions. One probe is labeled with a detectable marker andthe other probe is bindable on a support so that it can be captured by amembrane having a binding partner.

The prior art also describes bisulfite catalyzed transaminationreactions with cytidine.

Of the four nucleic acids, cytidine uniquely undergoes transaminationreaction in the presence of bisulfite.

Bisulfite modifications of nucleic acids is extensively discussed inProg. Nucl. Acid Res. Mol. Biol., 16: 75 (1976). "Bisulfite Modificationof Nucleic Acids and their Constituents," (Hikoya Hayatsu).

Nucleic Acid Research, Volume 12, Number 2, 1984, pg. 989, describes theattachment of reporter groups to specific, selected cytidine residues inRNA using a bisulfite catalyzed transamination reaction.

Biochemical and Biophysical Research Communications, Vol 142, No. 2,1987, Jan. 30, 1987, pp. 519, describes the linking of cytidine to abiotin hydrazide using a bisulfite catalyzed reaction.

Analytical Biochemistry, 157, 199-207 (1986), pp 199, describes N⁴(6-aminohexyl)cytidine and cytidine-containing nucleotides where thesecompounds are formed using a bisulfite catalyzed transaminationreactions.

Journal of the American Chemical Society, 95, 14, July 14, 1973, pp.4746, describes bisulfite catalyzed isotope exchange on cytidine.

Nucleic Acids Research, Vol. 9, Nov. 5, 1981, pp. 1203 coupling t-RNAwith N-hydroxysuccinimide by way of a bisulfite catalyzedtransamination.

J. Chem. Biol., 1979, 78(i), 61-75 describes the bisulfite catalyzedtransamination of cytidine and acylhydrazides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates attachment of DNA to a solid support by atransamination reaction.

FIG. 2A illustrates a specific binding sequence complementary to the DNAto be determined.

FIG. 2B illustrates a specific binding sequence complementary to asection of polynucleic acid which is not complementary to the DNA to bedetermined.

SUMMARY OF THE INVENTION

This invention encompasses diagnostic reagents comprising a polynucleicacid probe having a specific binding sequence and having a region of oneor more cytidines, generally a polycytidine, wherein any cytidines inthe specific binding sequence are replaced with 5-methylcytidine andwherein the polynucleic acid probe is bound to an aminofunctionalizedsolid support by a bisulfite mediated transamination reaction betweenthe cytidine and the amino group of the solid support.

The cytidines provide for site specific binding of the polynucleic acidprobe to the solid support. The cytidine region may be located at the 3'or 5' end of the specific binding sequence or the cytidine region may bean internal sequence having nucleic acid sequences on either side whereat least one of the side sequences preferably both are specific bindingsequences. Any cytidines in the specific binding sequence are replacedwith 5-methylcytidine The 5-methylcytidine does not interfere with thespecific binding but prevents bisulfite catalyzed transaminationreaction. This feature of the invention permits site specific binding ofthe cytidine region of the probe to the solid support.

The specific binding sequence of the polynucleic acid probe may becomplementary and directly bindable to the DNA or RNA sequence to bedetected or the specific binding sequence may be complementary to aportion of a nucleic acid sequence which is bound to the nucleic acidsequence to be determined. The specific binding sequence also includeshomopolymer sequences such as polyadenosine, polyguanosine,polythymidine and poly 5-methylcytidine.

The reagents of this invention are useful in a variety of testconfigurations for detecting genetic material from cells includingmammalian cells, viruses, bacteria, and fungi.

DETAILED DESCRIPTION OF THE INVENTION

The polynucleotide probes are made by conventional automated methods forsynthesizing polynucleic acids M. Caruthers, Genetic Engineering, pp.119-145, Plenum Press, New York and London (1982). These methods involvethe repetitive formation of an ester linkage between an activatedphosphoric acid function of one nucleotide and the hydroxyl group ofanother nucleotide forming a phosphodiester bridge The exception is thatthe cytidines in the specific binding sequence are replaced with5-methylcytidine. In one embodiment the polynucleic acid probeterminates at the 3' or 5' end with 1 to 100 cytidines, preferably 1 to10 cytidines. In another embodiment the cytidine region has nucleic acidsequences on both ends at least one of which is a specific bindingsequence. In this embodiment the polycytidine region is about 5 to 10cytidines. These probes are suitable for binding to an aminofunctionalized support by a bisulfite catalyzed transamination reaction.

The amino functionalized solid supports are well known. Aminofunctionalized magnetic beads of iron-oxide coated with organic polymerscontaining an amino group are sold by Advanced Magnetics, Inc . 61 MoonyStreet. Cambridge, Mass. 02138. Polystyrene microspheres with aminogroups are sold by Polysciences, Inc., 400 Valley Road, Warrington, Pa.18976.

Polystyrene supports can be conveniently functionalized by the acidcatalyzed reaction of the polystyrene with N-hydroxymethylphthalimidefollowed by treatment with hydrazine to provide an aminomethyl group onthe phenyl ring as illustrated in more detail in Example 1.

The polynucleic acid probe is bound to the amino functionalized supportby reaction in about 2 0 molar bisulfite solution typically.

The support may be in the form of titer plate wells, dip sticks, beads,filter membranes and the like. The only requirement is the availabilityof free amino groups for the bisulfite catalyzed transamination. Theamino groups can be on a polymer which is then coated on the solidsupport so that the amino groups are available for a transaminationreaction.

Scheme I illustrates binding nucleic acids to an amino functionalizedsupport as follows: ##STR1## Cytidines in the nucleic acid moiety arereplaced with 5-methylcytidine.

Scheme II illustrates a target capture of poly G by a poly5-methylcytidine probe. ##STR2##

Scheme III illustrates non-terminal cytidines reacting to produce"branched"60 nucleic acid structures on the solid support.

In Schemes I and II n is equal to or greater than zero but generally isa number that provides for 1 to 100 cytidines preferably 1 to 10. InScheme III n is generally between 5 and 10.

The specific binding sequence may be complementary to a segment of theDNA or RNA to be determined as illustrated in FIG. 2A or the specificbinding sequence may be complementary to a segment of a polynucleic acidwhere the polynucleic acid has another segment complementary to the DNAor RNA to be determined as illustrated by FIG. 2B.

The labeled probe may be obtained from messenger RNA. from cDNA obtainedby reverse transcription of messenger RNA with reverse transcriptase orby cleavage of the genome, conveniently by endonuclease digestion,followed by cloning of the gene or gene fragment in accordance withknown techniques. See, for example, Kornberg, DNA Replication, W. H.Freeman and Co., San Francisco, 1980, pp. 670-679;Dallas et al. supra;So et al., supra; So et al. Infect. Immun. 21:405-411, 1978. Afterisolation and characterization of the desired gene or DNA fragment, thegene or DNA fragment may be used for preparation of the labeled probe orcloned for production of messenger RNA, which may then be used forpreparation of the probe.

Enzymes of interest as labels will primarily be hydrolases, particularlyesterases and glycosidases, or oxidoreductases, particularlyperoxidases. Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.Chemiluminescers include luciferin, and 2,3-dihydrophthalazinediones,e.g. luminol Viral detection agents include bacteriophage, X 174 and M13.

The oligomer 3'-TTT TTT TTT TCC CCC-5was prepared on a DNA synthesizerby the phosphoramidite method. The oligomer was then radioactivelylabeled by the enzymatic addition of several ³ H-TTP residues usingterminal deoxynucleotidyl transferase A fine dispersion of the aminofunctionalized latex beads in aqueous 1M sodium bisulfite solution at pH7 was formed by sonication and the ³ H-DNA was then added. At intervals,aliquots of the bead suspension were removed, the beads isolated and theamount of DNA bound to the support was determined by scintillationcounting. The results are displayed graphically in FIG. 1. These resultsillustrate that the DNA oligomer has been attached to the support.

Table I further illustrates the time binding of DNA to the support.

                  TABLE I                                                         ______________________________________                                        TIME-COURSE OF C.sub.5 T.sub.10 BINDING TO BENZYLAMINE                        SUPPORT                                                                               dN.sub.50 Capacity (ng/mg)                                                          IA HSO.sub.3           IB                                                     [C.sub.5 T.sub.10 ] =                                                                    IIA HSO.sub.3                                                                             Control                                  Days .sup.32 P(dN).sub.50                                                                   10 D/ml    [C.sub.5 T.sub.10 ] = 20 D/ml                                                             (Phosphate)                              ______________________________________                                         1   A        30.91      23.38       15.25                                         T        0.97       0.11        0.10                                          A        <0.48>     <0.23>      <0.37>                                    3   A        171.18     169.93      11.70                                         T        0.31       0.70        0.11                                          A        <6.77>     <2.91>      <0.01>                                    5   A        167.37     204.25      7.40                                          T        1.17       0.12        0.25                                          A        <12.78>    <5.19>      <0.17>                                   12   A        231.97     182.99      21.82                                         T        0.30       0.55        1.10                                          A        <16.50>    <10.27>     <0.14>                                   20   A        180.08     263.01      8.12                                          T        0.13       0.08        0.24                                          A        <17.76>    <20.33>     <0.80>                                   ______________________________________                                         Numbers in < > indicate signal counts left on beads after elution step.  

Microorganisms which may be diagnosed include bacteria viruses, fungi,protozoa, molds etc Among toxin producing microorganisms are bacteria,such as gram negative bacilli e.g. Escherichia, Vibrio, Yersinia,Klebsiella and Salmonella Species include E coli, Vibrio cholerae,Haemophilus ducrei, Legionaire's bacillus. Other microorganisms ofinterest are those difficult to cultivate such as Chlamydia trachomatis.genital Herpes virus, HIV, Norwalk Agent, Rotavirus, Cytomegalovirus,Campylobacter jejuni.

Genetic material of mammalian cells such as oncogenes can likewise bedetected.

The cells are treated to liberate their DNA (and/or RNA). If the cellsare provided with nutrients to expand their numbers, after a sufficienttime for the colonies to form, the filter is removed from the nutrientsource and the cells lysed. Lysis conditions are devised such that thecells or colonies do not migrate and their DNA remains affixed in placeon the surface where they were situated. The lysing and DNA denaturingas well as the subsequent washings can be achieved by placing the filtercontaining the cells or colonies, isolate side up, onto a bibuloussupport saturated with an appropriate solution for a sufficient time tolyse the cells and denature the DNA. For lysing, chemical lysing willconveniently be employed, usually dilute aqueous alkali e g. 0.1 to 1 MNaOH. The alkali will also serve to denature the DNA. Other denaturationagents include, elevated temperatures, organic reagents, e.g. alcohols,amides, amines, ureas, phenols and sulfoxides or certain inorganic ions,e.g. thiocyanate and perchlorate.

Denatured DNA can be transferred to an amino functionalized filter paperhaving a polynucleic acid probe bound to it. The filter paper is washedand reacted with a label probe having a complementary sequence to adifferent area of the DNA or RNA to be detected. The preparation ofsamples for detecting salmonella are described in detail in U.S. Pat.No. 4.689.295 which is incorporated herein by reference.

A procedure for testing for bacteria in food involves culturing about 25grams of food in nutrient broth for about 24 hours at 30.37° C. A 1 mlaliquot of the culture is filtered so that bacteria will be collected onthe filter. The bacteria are then lysed and denatured with a NaOH/NaClsolution and the lysate is removed from the filter and neutralized withtris buffer The lysate is then incubated with a reagent of thisinvention, i.e. support with a polynucleic acid probe specific to thebacteria being tested. The reagent (support) is then separated andwashed with tris buffer to remove non-specifically bound material. Thereagent is then reacted with a labeled probe which specifically binds toa different region of the DNA being tested and washed The label on thesupport is then measured.

The invention is illustrated further by the following examples which arenot to be construed as limiting the invention in scope or spirit to thespecific procedures described in them.

EXAMPLE 1 Amino Functionalization of Phenyl Groups of Solid Support

To 10 g divinylbenzene polystyrene beads was added in sequence, 75 mldichloromethane, 75 ml trifluoroacetic acid, 15 gN-hydroxymethylphthalimide and finally 10 g trifluoromethylsulfonicacid. The resulting mixture was stirred at room temperature for sixteenhours and the beads were removed by filtration. The beads were thenwashed extensively with ethanol (1000 ml) water (1000 ml) and finallyethanol until the filtrate was neutral to pH paper. The beads were thentreated with a solution of 8ml hydrazine monohydrate and 32ml water in160ml ethanol and the resulting mixture was mechanically stirred at roomtemperature overnight. The beads were removed by filtration and washedextensively with ethanol (1000 ml), water (1000 ml) and finally ethanoluntil the washings no longer gave a coloration with picryl sulfonic acid(to ensure complete removal of the hydrazine). The beads were air driedand finally dried in vacuo at room temperature. This support was storedfor use in the transmination chemistry. The above reactions areillustrated by the following scheme. ##STR3##

EXAMPLE 2 Hybridization of Nucleotide to Solid Support A. Washing

A 2.5% (v/v) suspension of transaminated solid support in 20 mM sodiumphosphate solution, pH7, was prepared. After centrifugation anddecantation the solid support was washed 5 times with wash buffer 5XSSC. A stock of 20X SSC was prepared by adding 800ml of water to 175.3 gof sodium chloride 88.2g of sodium citrate, adjusting the pH to 7 with 1M HCl and adding water to a final volume of 11.

B. Prehybridization

A prehybridization buffer (PHB) was prepared from the followingcomponents. 3ml 10% (w/v) polyvinylpyrolidone, 3ml of 10% BSA 3ml of 10%(w/v) Ficoll 400 detergent, 2.2 ml salmon sperm DNA stock (10 mg/ml), 15ml 20× SSC buffer stock and 250 mg yeast tRNA was dissolved in water toa final volume of 150ml A final wash was carried out at 55° C. in 500μlof PHB for 1 hour, followed by washing twice at room temperature with 5XSSC. The solid support was then prehybridized at room temperature for 3hours with 500μl of PHB buffer and then again washed with wash buffer.

C. Hybridization

Hybridization was carried out in 500μl of PHB buffer to which had beenadded a radiolabelled polynucleotide, for example [³² P]dA₅₀ (50-100μg/ml) at room temperature for 1 hour and then centrifuging at 200rpm ona rotator. After centrifugation and removal of the supernatent the solidsupport was then washed 5 times with PHB.

D. Thermal Elution Of The Bound Oligonucleotide

The solid support was incubated at 65° C. for 5 minutes in 500μl of 1XSSC. After centrifugation, the supernatent was removed and added toscintillation vials. This process was repeated and the supernatent wasadded to the original supernatent. E. The radioactivity in thesupernatent and that still remaining on the solid support was determinedby scintillation counting.

Table 2 illustrates the binding capacity of a polycytidine probe tovarious target nucleotides.

                  TABLE 2                                                         ______________________________________                                        Binding Capacity (ng/mg)                                                      C(m.sup.5 C).sub.10 C-Support                                                         Solid Support Before                                                                        Solid Support After                                             65° C. Elution                                                                       Elution                                                 .sup.32 P(dN).sub.n                                                                     bisulfite                                                                              NaCl       bisulfite                                                                            NaCl                                     ______________________________________                                        .sup.32 P-A.sub.50                                                                      9.73     13.85      6.45   13.68                                    .sup.32 P-T.sub.50                                                                      2.33     2.38       1.33   1.18                                     .sup.32 P-C.sub.12-18                                                                   3.70     3.55       2.33   1.08                                     .sup.32 P-G.sub.12-18                                                                   152.50   26.48      151.90.sup.1                                                                         22.35                                    .sup.32 P-G.sub.12-18                                                                   148.85   21.20      112.93.sup.1                                                                         15.58                                    .sup.32 P-t-RNA                                                                         47.43    18.68      38.13  4.85                                     ______________________________________                                         .sup.32 PA.sub.50 is phosphorous 32 labeled polyadenosine of about 50         residues.                                                                     .sup.32 PT.sub.50 is phosphorous 32 labeled polythymidine of about 50         residues.                                                                     .sup.32 PC.sub.12-18 is phosphorous 32 labeled polycytidine of 12 to 18       residues.                                                                     .sup.32 PG.sub.12-18 is phosphorous 32 labeled polyguanosine of 12 to 18      residues.                                                                     .sup.32 Pt-RNA is phosphorous 32 labeled tRNA.                                .sup.1 These results indicate that the support in this case can               irreversibly bind .sup.32 PG.sub.12-18.                                  

EXAMPLE 3 Attachment of T₁₀ C₅ T₁₀ to Polystyrene Beads

Amino functionalized polystyrene beads (62 5mg) were suspended in 5ml ofa pH 7, 2.5M NaHSO₃ solution Any clumps of beads were broken up firstwith a spatula and further with sonication for about fifteen minutes.

A T₁₀ C₅ T₁₀ probe solution having 5 OD's/ml (61 ) was added to thesolution containing the polystyrene beads The mixture was placed on arotator and circulated for three days at room temperature. A time courseexperiment indicated that maximum nucleic acid attachment occurred afterthree days of these conditions.

The reaction solution was washed four times with 20mM phosphate/25%ethanol incubating in 5ml buffer at 52 to 55° C. for one hour (to removeany non-specifically binding probe) and washing four additional timeswith buffer. After the final buffer wash, the beads were suspended in2mL of buffer and stored in a refrigerator until used for hybridization.

Hybridization according to the procedures of Example 2 with A₅₀, T₅₀,and t-RNA are illustrated in the following Table 3.

                  TABLE 3                                                         ______________________________________                                        BINDING DATA                                                                                    NUCLEIC ACID                                                                  CAPACITY (ng/mg)                                            Support      .sup.32 P-N,A.                                                                           NaCl    NaHSO.sub.3 --                                ______________________________________                                        PS-T.sub.10 C.sub.5 T.sub.10                                                               A.sub.50   17.18   265.80                                        PS-T.sub.10 C.sub.5 T.sub.10                                                               T.sub.50   0.29    0.93                                          PS-T.sub.10 C.sub.5 T.sub.10                                                               t-RNA      2.39    2.34                                          ______________________________________                                         PS = polystyrene                                                              The use of T.sub.10 C.sub.5 T.sub.10 gives increased binding capacity of      35% relative to C.sub.5 T.sub.10.                                        

What is claimed is:
 1. A diagnostic reagent comprising a polynucleicacid probe having a specific binding sequence and having a cytidineregion of one or more cytidines outside of the specific binding sequenceand wherein cytidines in the specific binding sequence are5-methylcytidines, wherein the polynucleic acid probe is bound to anamino functionalized support by a bisulfite mediated transaminationbetween a cytidine outside the specific binding sequence and an aminogroup on the support.
 2. A diagnostic reagent according to claim 1wherein the specific binding sequence is complementary to the DNA or RNAto be detected.
 3. A diagnostic reagent according to claim 1 wherein thecytidine region is 1 to 100 cytidines at a terminal end of thepolynucleic acid probe.
 4. A diagnostic reagent according to claim 1wherein the cytidine region is about 5 to 10 cytidines and is not at theterminal end of the polynucleic acid probe.
 5. A diagnostic reagentaccording to claim 1 wherein the specific binding sequence is ahomopolymer sequence selected from polyadenosine, polyguanosine,polythymidine, or poly 5-methylcytidine.
 6. A diagnostic reagentaccording to claim 1 wherein the specific binding sequence binds toviral DNA or RNA.
 7. A diagnostic reagent according to claim 1 whereinthe specific biding sequence binds to bacterial DNA or RNA.
 8. Adiagnostic reagent according to claim 1 wherein the specific bindingsequence binds to mammalian DNA or RNA.
 9. A diagnostic reagentaccording to claim 1 wherein the specific binding sequence binds tofungal DNA or RNA.
 10. A diagnostic test kit comprising:(a) a diagnosticreagent of claim 1 wherein the specific binding sequence iscomplementary to one portion of the DNA or RNA to be determined, orwhich is complementary to a portion of a polynucleotide bound to a DNAor RNA to be determined and (b) a labeled polynucleic acid probe havinga specific binding sequence complementary to a second portion of the DNAor RNA to be determined.
 11. A polynucleic acid probe comprising aspecific binding sequence and containing outside of the specific bindingsequence a cytidine region of one or more cytidines and wherein thecytidines in the specific binding sequence are 5-methylcytidines.
 12. Apolynucleic acid probe according to claim 11 wherein the specificbinding sequence is complementary to the DNA to be determined.
 13. Apolynucleic acid probe according to claim 11 wherein the cytidine regionis at a terminal end of the polynucleic acid probe.
 14. A polynucleicacid probe according to claim 11 wherein the cytidine region is not at aterminal end of the polynucleic acid probe.
 15. A polynucleic acid probeaccording to claim 11 wherein the specific binding sequence is ahomopolymer sequence.
 16. A method for detecting the presence ofspecific bacteria in bacteria containing samples comprising:(a)providing a sample suspected of containing the specific bacteria to bedetected; (b) lysing the bacterial in said sample to release their DNA;(c) denaturing the DNA; (d) contacting the denatured DNA with adiagnostic reagent of claim 1 and allowing the DNA of the reagent tohybridize with the DNA of the bacteria to be detected; (e) washing thereagent to remove non-specifically bound material; (f) hybridizing alabeled probe to the bacteria DNA bound to the reagent; (g) washing thereagent to remove non-specifically bound DNA; and measuring the label onthe reagent.